BMP growth factors and Phox2 transcription factors can induce synaptotagmin I and neurexin I during sympathetic neuron development.

Synaptotagmin I and neurexin I mRNAs, coding for proteins involved in neurotransmitter secretion, become detectable in primary sympathetic ganglia shortly after initial induction of the noradrenergic transmitter phenotype. To test whether the induction of these more general neuronal genes is mediated by signals known to initiate noradrenergic differentiation in a neuronal subpopulation, we examined their expression in noradrenergic neurons induced by ectopic overexpression of growth and transcription factors. Overexpression of BMP4 or Phox2a in vivo results in synaptotagmin I and neurexin I expression in ectopically located noradrenergic cells. In vitro, BMP4 initiates synaptotagmin I and neurexin I expression in addition to tyrosine hydroxylase induction. Thus, the induction of synaptotagmin I and neurexin I, which are expressed in a large number of different neuron populations, can be accomplished by growth and transcription factors available only to a subset of neurons. These findings suggest that the initial expression of proteins involved in neurotransmitter secretion is regulated by different signals in different neuron populations.

Ligands and signaling through receptor-type tyrosine phosphatases.

Virtually every aspect of cellular proliferation and differentiation is regulated by changes in tyrosine phosphorylation. Tyrosine phosphorylation, in turn, is controlled by the opposing activities of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). PTKs are often transmembrane proteins (receptor PTKs) whose enzymatic activities and signaling functions are tightly regulated by the binding of specific ligands. A variety of transmembrane PTPs has also been identified; these proteins are called receptor PTPs (RPTPs), but in most cases their roles as receptors are very poorly understood. This review discusses the evidence that RPTPs are actually receptors for extrinsic ligands, and the extent to which interactions with putative ligands are known or suspected to cause changes in enzymatic activity. Finally, some of the RPTP substrates believed to be physiologically important are described. The evidence gathered to date suggests that models derived from studies of receptor PTKs may be too simple to account for the diversity and complexity of mechanisms through which ligand binding controls RPTP function.

CRYP-2/cPTPRO is a neurite inhibitory repulsive guidance cue for retinal neurons in vitro.

Receptor protein tyrosine phosphatases (RPTPs) are implicated as regulators of axon growth and guidance. Genetic deletions in the fly have shown that type III RPTPs are important in axon pathfinding, but nothing is known about their function on a cellular level. Previous experiments in our lab have identified a type III RPTP, CRYP-2/cPTPRO, specifically expressed during the period of axon outgrowth in the chick brain; cPTPRO is expressed in the axons and growth cones of retinal and tectal projection neurons. We constructed a fusion protein containing the extracellular domain of cPTPRO fused to the Fc portion of mouse immunoglobulin G-1, and used it to perform in vitro functional assays. We found that the extracellular domain of cPTPRO is an antiadhesive, neurite inhibitory molecule for retinal neurons. In addition, cPTPRO had potent growth cone collapsing activity in vitro, and locally applied gradients of cPTPRO repelled growing retinal axons. This chemorepulsive effect could be regulated by the level of cGMP in the growth cone. Immunohistochemical examination of the retina indicated that cPTPRO has at least one heterophilic binding partner in the retina. Taken together, our results indicate that cPTPRO may act as a guidance cue for retinal ganglion cells during vertebrate development.

Growth cone steering by receptor tyrosine phosphatase delta defines a distinct class of guidance cue.

Receptor-type tyrosine phosphatases (RPTPs) are involved in pathfinding decisions by elongating axons, but how they function in these decisions remains unclear. A vertebrate RPTP, PTP-delta, is a neurite-promoting homophilic adhesion molecule; here we demonstrate chemoattraction of CNS growth cones by a locally applied gradient of soluble PTP-delta. The attractive effect of PTP-delta was abolished by inhibition of tyrosine phosphatase activity, but in contrast to other guidance proteins was unaffected by inhibition of cyclic nucleotide activities. Gradients of PTP-delta or of laminin-1 also promoted increases in the speed of growth cone migration, but laminin-1 did not steer growth cones. Our results indicate that PTP-delta is a chemoattractant for vertebrate CNS neurons in vitro and suggest that it represents a distinct class of guidance protein from those previously defined. Further, our data indicate that growth cone attraction is mechanistically distinct from increases in the speed of growth cone movement.

Receptor tyrosine phosphatases in axon growth and guidance.

The last 5 years has seen an explosion of evidence linking RPTPs to the regulation of axon growth and guidance. Important questions to be addressed include the ligand-receptor interactions involved in axon growth regulation, the signaling pathways controlled by RPTPs in neurons, and the manner in which different RPTPs within a class, and different classes of RPTPs, coordinate their functions to ensure appropriate axon growth. Are RPTPs signaling ligands, signaling receptors, or both? Do RPTPs function mainly by modifying adhesive preferences, or are they instructive in guidance decisions? Do specific types of RPTPs send specific signals to neurons, or do they work together to fine-tune levels of tyrosine phosphorylation? Whatever the outcome, it seems certain that the answers to these questions will come only from a combination of the powerful genetic approaches possible in Drosophila (and in mice) with the biochemical and cell biological approaches possible in the vertebrate systems.

Tissue specific expression of Yrk kinase: implications for differentiation and inflammation.

The Src family of proto-oncogenes is a highly conserved group of non-receptor tyrosine kinases with very similar, but not identical, tissue distributions and functions. Yrk is a recently discovered new member of this family. Here we report the patterns of expression of this kinase in a variety of chicken tissues during development and after hatching, and experiments that correlate some of the observed patterns of expression with potential functions. The results show that the Yrk protein is primarily found in neuronal and epithelial cells and in monocyte/macrophages. In neuronal tissues of hatched chicks, Yrk is expressed in Purkinje cells, in the gigantocellularis of the brain-stem, and in retinal ganglion cells. In addition, staining for this kinase is also seen as thread-like and punctate patterns suggesting staining in neurites and growth cones. Epithelial cells express Yrk in the stomach during late developmental stages and after hatching but, in other epithelia such as in the peridermis, intestine and kidney, expression is high during development but low (skin) or undetectable (intestine and kidney) after hatching. These results suggest that Yrk may have several functional roles, specifically in cell migration and or differentiation during neuronal and epithelial cell development and in maintenance of the differentiated phenotype. In this study we also show that significant levels of Yrk are detected in monocytes of the blood and in tissue macrophages. Analysis of chicken hematopoietic cell lines confirmed the expression of Yrk in cells of monocyte/macrophage lineage and show for the first time in experimentally-induced inflammation that Yrk kinase activity is high during the period of monocyte infiltration, raising the possibility that this kinase plays a role in inflammation and/or response to injury.

Regulation of retinal neurite growth by alterations in MAPK/ERK kinase (MEK) activity.

Activation of the extracellular-signal regulated kinase (ERK) cascade may be involved in the promotion of neurite outgrowth by a variety of stimuli. For example, we have previously shown that laminin (LN) and N-cadherin activate ERK2 in chick retinal neurons, and that pharmacological inhibition of MAPK/ERK kinase (MEK), the major upstream ERK2 activator, severely impairs neurite growth induced by these proteins. We have therefore hypothesized that ERK activation through MEK is required for optimal induction of neurite growth by these proteins. Here we show that expression of mutant MEK in transfected retinal neurons alters neuronal responses to LN in a manner consistent with this hypothesis. Neurons expressing a constitutively active MEK construct extended longer neurites on LN than controls, while neurons transfected with a dominant negative construct extended shorter neurites. Further, experiments in which transfected neurons were replated onto polylysine substrates suggest that activation of MEK is sufficient for neurite promotion on a non-inducing substrate, and neurons replated onto LN confirm the pharmacological data that inhibition of MEK activation inhibits LN-induced neurite growth. We conclude that ERK activation plays a direct role in the promotion of neurite outgrowth from retinal neurons by LN.

Receptor tyrosine phosphatase-delta is a homophilic, neurite-promoting cell adhesion molecular for CNS neurons.

Appropriate regulation of tyrosine phosphorylation is essential for axon growth and guidance; evidence from invertebrates indicates that receptor-type tyrosine phosphatases (RPTPs) are required for correct axon growth during CNS development. One vertebrate RPTP, PTP-delta, is highly expressed in brain and has a cell adhesion molecule-like extracellular domain (ECD) comprising three immunoglobulin repeats and eight fibronectin type III repeats. Using fluorescent beads (Covaspheres) coated with the PTP-delta ECD, as well as insect cells expressing PTP-delta on their surfaces, we show that PTP-delta is a homophilic cell adhesion molecule. A variety of chick neurons adhere strongly to an Fc fusion protein containing the PTP-delta ECD. Additionally, substrate-bound PTP-delta ECD fusion protein strongly promotes neurite outgrowth from forebrain neurons; this effect is separable from its effect on adhesion. Our results indicate that PTP-delta is a neurite-promoting cell adhesion molecule for CNS neurons.

Tetraspanins expressed in the embryonic chick nervous system.

Proteins of the tetraspanin superfamily participate in the formation of plasma membrane signaling complexes; recent evidence implicates neuronal tetraspanins in axon growth and target recognition. We used a degenerate PCR screen to identify cDNAs encoding tetraspanins expressed in the embryonic spinal cord. Two cDNAs identified apparently represent chick homologues of NAG-2 (cnag) and CD9 (chCD9). A third clone encodes a novel tetraspanin (neurospanin). All three mRNAs are widely expressed but exhibit developmentally distinct patterns of expression in the nervous system. Both neurospanin and cnag exhibit high relative expression in nervous tissue, including brain, spinal cord and dorsal root ganglia (DRG).

The receptor tyrosine phosphatase CRYPalpha promotes intraretinal axon growth.

Retinal ganglion cell axons grow towards the optic fissure in close contact with the basal membrane, an excellent growth substratum. One of the ligands of receptor tyrosine phosphatase CRYPalpha is located on the retinal and tectal basal membranes. To analyze the role of this RPTP and its ligand in intraretinal growth and guidance of ganglion cell axons, we disrupted ligand- receptor interactions on the retinal basal membrane in culture. Antibodies against CRYPalpha strongly reduced retinal axon growth on the basal membrane, and induced a dramatic change in morphology of retinal growth cones, reducing the size of growth cone lamellipodia. A similar effect was observed by blocking the ligand with a CRYPalpha ectodomain fusion protein. These effects did not occur, or were much reduced, when axons were grown either on laminin-1, on matrigel or on basal membranes with glial endfeet removed. This indicates that a ligand for CRYPalpha is located on glial endfeet. These results show for the first time in vertebrates that the interaction of a receptor tyrosine phosphatase with its ligand is crucial not only for promotion of retinal axon growth but also for maintenance of retinal growth cone lamellipodia on basal membranes.

Distinct neurite outgrowth signaling pathways converge on ERK activation.

Several distinct classes of proteins positively regulate axonal growth; some of these are known to activate the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling cascade, at least in nonneuronal cells. We have found that N-cadherin, as well as laminin (LN) and basic fibroblast growth factor (bFGF), can activate ERK in embryonic chick retinal neurons. Additionally, adhesion of retinal neurons to LN or N-cadherin substrates induced a redistribution of ERK from the cytoplasm toward the plasma membrane. Neurite outgrowth induced by bFGF, LN, or N-cadherin was strongly inhibited by treatment with inhibitors of ERK kinase activation, but not by an inhibitor of p38 MAPK. We conclude (1) that N-cadherin and LN can activate ERK in retinal neurons and (2) that activation of ERK is required for full neurite outgrowth induced by these proteins. Our results suggest that ERK activation is one point of convergence for signaling pathways generated by a variety of axon growth inducers.

Expression of receptor tyrosine phosphatases during development of the retinotectal projection of the chick.

Receptor tyrosine kinases and receptor protein tyrosine phosphatases (RPTPs) appear to coordinate many aspects of neural development, including axon growth and guidance. Here, we focus on the possible roles of RPTPs in the developing avian retinotectal system. Using both in situ hybridization analysis and immunohistochemistry, we show for the first time that five RPTP genes--CRYPalpha, CRYP-2, PTPmu, PTPgamma, and PTPalpha--have different but overlapping expression patterns throughout the retina and the tectum. PTPalpha is restricted to Muller glia cells and radial glia of the tectum, indicating a possible function in controlling neuronal migration. PTPgamma expression is restricted to amacrine neurons. CRYPalpha and CRYP-2 mRNAs in contrast are expressed throughout the retinal ganglion cell layer from where axons grow out to their tectal targets. PTPmu is expressed in a subset of these ganglion cells. CRYPalpha, CRYP-2, and PTPmu proteins are also localized in growth cones of retinal ganglion cell axons and are present in defined laminae of the tectum. Thus, the spatial and temporal expression of three distinct RPTP subtypes--CRYPalpha, CRYP-2, and PTPmu--are consistent with the possibility of their involvement in axon growth and guidance of the retinotectal projection.

Fibroblast growth factor receptor signaling in Xenopus retinal axon extension.

Fibroblast growth factor receptors (FGFRs) and N-cadherin both regulate axon extension in developing Xenopus retinal ganglion cells (RGCs). Cultured cerebellar neurons have been shown to require FGFR activity for N-cadherin-stimulated neurite outgrowth, raising the possibility that N-cadherin is a FGFR ligand. To investigate this possibility in the developing visual system, retinal neurons were transfected with a dominant-negative FGFR (XFD) and plated on purified N-cadherin substrates. XFD-expressing neurons extended markedly shorter processes than control GFP-expressing neurons, implicating a role for FGFRs in N-cadherin-stimulated neurite outgrowth. To examine whether N-cadherin and FGFRs share the same pathway or use distinct second messenger pathways, specific inhibitors of implicated signaling molecules were added to neurons stimulated by N-cadherin, basic fibroblast growth factor (bFGF), or brain-derived nerve factor (BDNF) (which stimulates RGC outgrowth by a FGFR-independent mechanism). Diacylglycerol (DAG) lipase and Ca2+/calmodulin kinase II inhibitors both significantly reduced outgrowth stimulated by N-cadherin or bFGF but not by BDNF. Furthermore, we show that inhibiting DAG lipase activity in RGC axons extending in vivo toward the optic tectum reversibly slows axon extension without collapsing their growth cones. Thus, a common second-messenger signaling pathway mediating both N-cadherin- and bFGF-stimulated neurite extension is consistent with a model in which N-cadherin directly modulates the FGFR or a model whereby both FGFR and N-cadherin regulate the same second-messenger system.

Differential regulation of synaptic vesicle protein genes by target and synaptic activity.

Differentiation of presynaptic nerve terminals involves changes in gene expression; these may be regulated by synaptic transmission and/or by contact with the target muscle. To gain insight into the control of presynaptic differentiation, we examined the regulation by target and synaptic activity of synaptic vesicle protein (SVP) genes in the chick ciliary ganglion (CG). In the CG, two SVP genes, synaptotagmin I (syt I) and synaptophysin II (syp II), are coordinately upregulated at the time of target contact. To test the hypothesis that this upregulation is induced by target contact, we examined mRNA levels of syt I and syp II in CGs from embryos in which one eye had been removed before axon outgrowth. As expected, target removal prevented the normal upregulation of syt I mRNA in the deprived ganglion. In contrast, and unexpectedly, syp II mRNA upregulation was not affected. The target dependence of syt I upregulation was not attributable to nerve-muscle transmission, because blockade of this transmission had no effect on SVP mRNA levels. Surprisingly, blockade of synapses onto CG neurons from the brain also did not affect syt I mRNA levels but increased levels of syp II mRNA. We conclude that contact with target induces upregulation of syt I mRNA, which is the case for spinal motor neurons. However, the normal upregulation of syp II mRNA is not controlled by the same signal(s). Instead, our results suggest that these two SVP genes are differentially regulated, both by target contact and by blockade of synaptic transmission.

Agrin orchestrates synaptic differentiation at the vertebrate neuromuscular junction.

The synapse is a key structure that is involved in perception, learning and memory. Understanding the sequence of steps that is involved in establishing synapses during development might also help to understand mechanisms that cause changes in synapses during learning and memory. For practical reasons, most of our current knowledge of synapse development is derived from studies of the vertebrate neuromuscular junction (NMJ). Several lines of evidence strongly suggest that motor axons release the molecule agrin to induce the formation of the postsynaptic apparatus in muscle fibers. Recent advances implicate proteins such as dystroglycan, MuSK, and rapsyn in the transduction of agrin signals. Recently, additional functions of agrin have been discovered, including the upregulation of gene transcription in myonuclei and the control of presynaptic differentiation. Agrin therefore appears to play a unique role in controlling synaptic differentiation on both sides of the NMJ.

Extracellular-matrix molecules expressed by innervatable muscle.

Embryonic and adult muscle express distinct complements of proteins. Transplant experiments have shown that embryonic and denervated muscles can form synapses with foreign neurites, while normal innervated mature muscles cannot, except at the synaptic area. To identify molecular differences between 'innervatable' embryonic muscle and 'non-innervatable' mature muscle, we used a differential immunization method to make monoclonal antibodies that recognize antigens present in embryonic muscle rather than normal mature muscle. Four independent monoclonal antibodies that stain embryonic and mature muscle sections differentially have been obtained. One stains the entire embryonic muscle cell surface but only the synaptic area in mature muscle; 3 stain the entire embryonic muscle cell surface but do not stain mature muscle. The antibodies also stain other embryonic tissues at several stages. The antigens are concentrated in basal laminae and in an extracellular-matrix (ECM) fraction, indicating that they are ECM molecules. The temporal expression of all 4 antigens in developing muscle is coincident with muscle innervation. All antigens exhibit temporal and tissue distributions different from those of any reported muscle proteins, suggesting that novel antigens underlie these patterns. These results confirm that the immature muscle ECM has a distinct molecular composition, and suggest that the presence of these antigens may be part of the molecular basis for the innervatability of embryonic muscle.

Target contact regulates expression of synaptotagmin genes in spinal motor neurons in vivo.

During neuromuscular development, neuronal contact with peripheral targets is associated with an increase in synaptic vesicle protein (SVP) gene expression, suggesting that target contact and upregulation of SVP genes are causally related. To test this idea, we analyzed the developmental expression pattern of synaptotagmin (syt) mRNAs in the chick lateral motor column (LMC) using in situ hybridization. Syt I mRNA in the LMC is upregulated from Embryonic Day 4.5 (E4.5) to E5.5, coincident with the time these neurons begin to make contact with their muscle targets. In contrast, levels of mRNA for neurofilament do not change during this time. Extirpation of the limb bud prior to motor axon outgrowth eliminates the increase in syt I mRNA ipsilaterally. Later in development, there is a switch in syt isoform abundance in the LMC, with syt II mRNA being upregulated between E15 and E20 and syt I mRNA being downregulated. Our results suggest that contact with targets upregulates syt I gene expression during neuromuscular synapse formation in vivo, and that a later stage of synaptic maturation involves changes in SVP isoform abundance.

Evidence that agrin directly influences presynaptic differentiation at neuromuscular junctions in vitro.

The synaptic protein agrin is required for aspects of both pre- and postsynaptic differentiation at neuromuscular junctions. Although a direct effect of agrin on postsynaptic differentiation, presumably through the MuSK receptor, is established, it is not clear whether agrin directly affects the presynaptic nerve. To provide evidence on this point, we used anti-agrin IgG to disrupt agrin function in chick ciliary ganglion (CG) neuron/myotube cocultures. In cocultures grown in the presence of 200 microg/ml anti-agrin IgG, clustering of acetylcholine receptors (AChRs), extracellular matrix proteins, and the synaptic vesicle protein synaptotagmin (syt) at nerve-muscle contacts was inhibited. Syt clustering was still inhibited in the presence of 100 microg/ml blocking antibody, while the postsynaptic clustering of AChRs, heparan sulphate proteoglycan, and s-laminin was retained. Additionally, in CG neurons cultured with COS cells expressing agrin A0B0, which lacks the ability to signal postsynaptic differentiation, syt clustering was induced and this clustering was also blocked by anti-agrin IgG. Our results demonstrate that agrin function is acutely required for pre- and postsynaptic differentiation in vitro, and strongly suggest that agrin is directly involved in the induction of presynaptic differentiation.

CRYP-2: a receptor-type tyrosine phosphatase selectively expressed by developing vertebrate neurons.

Axonal growth and guidance, like other aspects of neuronal differentiation, can be regulated by changes in tyrosine phosphorylation. Although much is known concerning the role of tyrosine kinases in these processes, relatively little is known about the nature and function of protein tyrosine phosphatases (PTPs) that may be involved. To identify the PTPs expressed in the embryonic chicken CNS at the time of axon growth, we performed a polymerase chain reaction based "screen" using degenerate primers directed against conserved regions of the PTP catalytic domain. We obtained five distinct PTP-related cDNAs, two of which code for novel PTPs. One, designated CRYP-2, is selectively expressed in the CNS. Full-length cloning of CRYP-2 revealed that it is a receptor-type PTP with an adhesion molecule-like extracellular region comprising fibronectin (FN) type III repeats and a single catalytic domain in the intracellular region. It is alternatively spliced in the juxtamembrane region, similar to other PTPs recently cloned. CRYP-2 mRNA is strongly expressed in the brain during the time of axon growth; it is downregulated toward the end of embryogenesis. Western blot analysis identifies a 330-kDa glycoprotein as CRYP-2 and confirms that the protein is downregulated after hatching. Immunostaining of cerebellar neurons in vitro reveals that CRYP-2 is expressed on neuronal cell bodies and processes, but not on glia. The CAM-like structure, developmental pattern of expression, and neuron-specific localization of the CRYP-2 PTP suggest that it is involved in neuronal differentiation, particularly axon growth.

Intracellular mechanisms of axon growth induction by CAMs and integrins: some unresolved issues.

Integrins and cell adhesion molecules (CAMs) are important neuronal receptors mediating substrate-induced axon growth. Signaling of axon growth through these receptors involves both regulation of tyrosine phosphorylation and transient increases in intracellular Ca2+. Many of the details concerning these signal transduction events and mechanisms through which they regulate effectors of axon growth are poorly understood. This review discusses some of the gaps in our current knowledge, with suggestions on approaches to closing these gaps. Emphasis is on the role of tyrosine phosphatases in the regulation of axon growth, the origin and nature of Ca2+ signals produced by stimulation of CAMs and integrins, and possible links of these two pathways to cytoskeletal rearrangements and directed addition of plasma membrane.